1
00:00:01 --> 00:00:04
Good morning, class.
Nice to see you here,
2
00:00:04 --> 00:00:08
you loyal holdouts, the stalwarts
who haven't gone home early for
3
00:00:08 --> 00:00:12
Thanksgiving. You recall that
last time we were talking about the
4
00:00:12 --> 00:00:16
Matevoidic system, and
much of the rationale for
5
00:00:16 --> 00:00:20
studying it stems from two reasons.
First of all, it recapitulates in a
6
00:00:20 --> 00:00:25
formal sense what happens
during embryogenesis,
7
00:00:25 --> 00:00:29
i.e. one has relatively
undifferentiated stem cells which
8
00:00:29 --> 00:00:33
are able to differentiate into a
number of different directions by
9
00:00:33 --> 00:00:37
committing themselves to either
the myeloid or lymphoid compartment,
10
00:00:37 --> 00:00:41
and then going down yet other
pathways, more detailed pathways to
11
00:00:41 --> 00:00:46
generate a whole
variety of cell types.
12
00:00:46 --> 00:00:50
Secondly, we really understand
the differentiation pathways of
13
00:00:50 --> 00:00:54
Matevoisis better than we
understand any tissue in the body,
14
00:00:54 --> 00:00:59
in no small part because it's much
easier to study the soluble cells in
15
00:00:59 --> 00:01:03
the blood and in the immune system
than it is to study how these
16
00:01:03 --> 00:01:08
processes happen in normal
tissues. But having said that,
17
00:01:08 --> 00:01:13
I want to emphasize the fact that
in each of our tissues there are
18
00:01:13 --> 00:01:18
oligopotential stem cells.
When I say oligopotential I mean
19
00:01:18 --> 00:01:24
they can go down several different
pathways. Recall up there on that
20
00:01:24 --> 00:01:29
diagram we talked about
pluripotential which means multiple,
21
00:01:29 --> 00:01:34
and today we're going to talk a
bit about todipotential stem cells,
22
00:01:34 --> 00:01:39
which are able to disperse
descendants into all the different
23
00:01:39 --> 00:01:45
differentiation
lineages in the body.
24
00:01:45 --> 00:01:50
At the end of our last lecture,
we were focusing on the red blood
25
00:01:50 --> 00:01:56
cells. And this is sometimes
called erythropoiesis,
26
00:01:56 --> 00:02:02
which is to say the process by
which red blood cells are generated.
27
00:02:02 --> 00:02:06
We mentioned the concept of
homeostasis, and homeostasis just
28
00:02:06 --> 00:02:11
refers to the fact that all of these
systems are in very delicate balance
29
00:02:11 --> 00:02:15
so that the body can respond to the
physiologic needs of the organism at
30
00:02:15 --> 00:02:20
any one point in time. We
talked about the fact that for
31
00:02:20 --> 00:02:24
example when there's a
massive infection in the body,
32
00:02:24 --> 00:02:29
then the homeostatic mechanisms
allow an increase in these kinds of
33
00:02:29 --> 00:02:34
immune cells in order to
encounter the infection.
34
00:02:34 --> 00:02:38
And at the end of our last lecture,
we were talking about this specific
35
00:02:38 --> 00:02:43
branch, and how in fact
homeostasis is maintained there.
36
00:02:43 --> 00:02:48
And what we see here is a
series of committed progenitors.
37
00:02:48 --> 00:02:52
So when I talk about committed
progenitors I'm referring to cells
38
00:02:52 --> 00:02:57
that have already made the
commitment to go down one
39
00:02:57 --> 00:03:02
or another pathway. They're
not yet fully differentiated.
40
00:03:02 --> 00:03:06
As you can see here, we
have first forming cells and
41
00:03:06 --> 00:03:10
colony forming cells. We
don't need to remember all the
42
00:03:10 --> 00:03:14
different abbreviations except to
say that these cells here are in a
43
00:03:14 --> 00:03:18
relative undifferentiated
state. And the only end stage
44
00:03:18 --> 00:03:22
differentiation comes at the very
end here when we get to red blood
45
00:03:22 --> 00:03:26
cells. We said in general that
it's the case that most highly
46
00:03:26 --> 00:03:30
differentiated cells are
post-mitotic, which is to say
47
00:03:30 --> 00:03:34
they're never going to reenter into
the growth and division cycle of the
48
00:03:34 --> 00:03:38
cell that we talked about
earlier in the semester.
49
00:03:38 --> 00:03:42
And that's obviously dictated here
by the fact that this erythrocyte
50
00:03:42 --> 00:03:46
lacks a nucleus, i.e.
during the final stage of
51
00:03:46 --> 00:03:50
differentiation, in addition
to accumulating large
52
00:03:50 --> 00:03:54
amounts of hemoglobin in its
cytoplasm, this cell actually pops
53
00:03:54 --> 00:03:58
out its nucleus, and that
obviously represents an
54
00:03:58 --> 00:04:02
irrevocable change in that cell
can never again enter into growth
55
00:04:02 --> 00:04:06
and division cycle. The
immediate precursor of an
56
00:04:06 --> 00:04:12
erythrocyte is often called an
erythroblast. And the term blast
57
00:04:12 --> 00:04:17
here refers to a cell of embryonic
appearance. Blast is used often to
58
00:04:17 --> 00:04:23
indicate, we'll mention that again
shortly, a cell which looks very
59
00:04:23 --> 00:04:28
primitive, and embryonic, and
undifferentiated. And that ends
60
00:04:28 --> 00:04:34
up going into an erythrocyte,
which we said is actually a synonym
61
00:04:34 --> 00:04:40
for a red blood cell,
an RBC, a red blood cell.
62
00:04:40 --> 00:04:44
And we talked about the fact
that this progression is actually
63
00:04:44 --> 00:04:49
maintained and furthered by the
stimulus of the compound called
64
00:04:49 --> 00:04:53
erythropoietin. So, we're
using some of the same
65
00:04:53 --> 00:04:58
words over and over again. And
erythropoietin is essentially a
66
00:04:58 --> 00:05:03
growth factor which stimulates the
end stage differentiation of the
67
00:05:03 --> 00:05:08
erythroblast into
the erythrocyte.
68
00:05:08 --> 00:05:13
Epo, as erythropoietin's often
abbreviated, is actually made in the
69
00:05:13 --> 00:05:19
kidneys. And it's made in
the kidneys in response to the
70
00:05:19 --> 00:05:25
physiological stimulus of
hypoxia. Hypoxia means inadequate
71
00:05:25 --> 00:05:31
oxygenation of the tissues.
You might ask, well, why is red
72
00:05:31 --> 00:05:37
blood cell contractions controlled,
as they are, in the kidney?
73
00:05:37 --> 00:05:41
And the fact is, we don't
really know why evolution
74
00:05:41 --> 00:05:45
has chosen the kidney as the site of
monitoring the degree of oxygenation
75
00:05:45 --> 00:05:49
of the blood. And in response to
hypoxia, it begins to crank out
76
00:05:49 --> 00:05:53
erythropoietin, or
Epo. You can think of
77
00:05:53 --> 00:05:57
erythropoietin as an extracellular
liggon just like a growth factor.
78
00:05:57 --> 00:06:01
It has its own cognate receptor
on the surface of the erythroblast,
79
00:06:01 --> 00:06:06
and when Epo released by the kidney
hits an erythroblast in the context
80
00:06:06 --> 00:06:11
of the bone marrow, it
actually has two effects.
81
00:06:11 --> 00:06:16
It happens to be the case that
roughly even 95% of the erythroblast
82
00:06:16 --> 00:06:21
that are made routinely are forced
to go into apitosis under routine
83
00:06:21 --> 00:06:26
conditions. So, this is
an enormously wasteful
84
00:06:26 --> 00:06:31
system, i.e. as every moment we
speak, 90 or 95% of the erythroblast
85
00:06:31 --> 00:06:37
that have come into existence
in your bone marrow apitose.
86
00:06:37 --> 00:06:43
They never go into end
stage differentiation.
87
00:06:43 --> 00:06:50
But when Epo is around, Epo
provides a strong anti-apoptotic
88
00:06:50 --> 00:06:56
signal to the red blood saves
some and maybe even all of the
89
00:06:56 --> 00:07:03
erythroblasts from their normal
fate of undergoing apitosis.
90
00:07:03 --> 00:07:07
So here, if we imagine
there are actually two fates,
91
00:07:07 --> 00:07:12
one is to become an erythrocyte,
and the other is to apitose, where
92
00:07:12 --> 00:07:16
the aptisosis is paradoxically
enough the dominant fate of the cell,
93
00:07:16 --> 00:07:21
the moment that an Epo comes on the
scene, it blocks this alternative
94
00:07:21 --> 00:07:25
fate, allowing these cells to mature.
Epo at the same time stimulates the
95
00:07:25 --> 00:07:30
erythroblast to differentiate.
Now, you might as yourself the
96
00:07:30 --> 00:07:35
question, why is there this
enormously inefficient process?
97
00:07:35 --> 00:07:38
An enormous effort is made to crank
out large, astronomical numbers of
98
00:07:38 --> 00:07:42
erythroblasts, and yet
most of them are wasted even
99
00:07:42 --> 00:07:46
before they've had a chance to
undergo end stage differentiation.
100
00:07:46 --> 00:07:50
And the rationale here is as
follows. This is a terrific system
101
00:07:50 --> 00:07:54
for rapidly ramping up the level of
red blood cells in your circulation
102
00:07:54 --> 00:07:58
because here, within a matter of
a day or two, one can crank up,
103
00:07:58 --> 00:08:02
actually in a matter of hours,
you can crank up the rate of
104
00:08:02 --> 00:08:06
production of red blood cells
by maybe even a factor of ten.
105
00:08:06 --> 00:08:10
Instead of having 90% of
the erythroblast apitose,
106
00:08:10 --> 00:08:14
let's say 0% of them do so, and
therefore, instead of having 10%
107
00:08:14 --> 00:08:18
of the erythroblasts becoming red
blood cells, 100% of them will do so.
108
00:08:18 --> 00:08:22
And therefore, you have
the virtually miraculous
109
00:08:22 --> 00:08:26
response that if you go from here
high up in the rocky mountains at
110
00:08:26 --> 00:08:30
ten or 12,000 feet, within a
matter of two or three days,
111
00:08:30 --> 00:08:34
your red blood cell concentration
actually has compensated,
112
00:08:34 --> 00:08:38
has risen up to create the oxygen
caring capacity that enables you to
113
00:08:38 --> 00:08:42
deal with the thin oxygen, with
the low oxygen tension that's
114
00:08:42 --> 00:08:47
present at high altitudes.
Now, having said that,
115
00:08:47 --> 00:08:52
the fact is that there is an Epo
receptor on the surface of the
116
00:08:52 --> 00:08:58
erythroblast, and what we
see there is the following.
117
00:08:58 --> 00:09:01
Let's talk about the erythroblast
and just blow it up a little bit.
118
00:09:01 --> 00:09:05
So, here's the erythroblast.
That's the undifferentiated
119
00:09:05 --> 00:09:08
precursor. And by the way, the
erythroblast is actually still a
120
00:09:08 --> 00:09:12
white blood cell. Often we
call a white blood cell a
121
00:09:12 --> 00:09:15
leukocyte. You may know
that gluco means white. So,
122
00:09:15 --> 00:09:19
a leukocyte, it's still white.
And after the erythropotent
123
00:09:19 --> 00:09:22
impinges on it, one of the
things it starts doing is
124
00:09:22 --> 00:09:26
to make the hemoglobin, which
turns it into a red blood cell.
125
00:09:26 --> 00:09:30
At this stage, it's still white.
On the surface of the erythroblast
126
00:09:30 --> 00:09:35
are these Epo receptors. I'll
just abbreviate them like this,
127
00:09:35 --> 00:09:40
Epo receptor, and once it binds
the liggon Epo just like the growth
128
00:09:40 --> 00:09:45
factor receptors, we talked
early in the receptors
129
00:09:45 --> 00:09:49
signals are sent into the
erythroblast to stimulate both
130
00:09:49 --> 00:09:54
differentiation and to prevent
the initiation of the cell suicide
131
00:09:54 --> 00:09:59
program that we call apitosis.
Interestingly, one of the things
132
00:09:59 --> 00:10:04
that happens normally is the
following, that when these signals
133
00:10:04 --> 00:10:09
come in, there is an enzyme called
a phosphotase which is attracted
134
00:10:09 --> 00:10:14
to the receptor. The
Epo receptor works like a
135
00:10:14 --> 00:10:18
tyrosine kinase growth factor
receptor that we talked about
136
00:10:18 --> 00:10:22
earlier in the semester.
And here, we have an enzyme,
137
00:10:22 --> 00:10:27
a phosphotase, which actually
counteracts the function of the
138
00:10:27 --> 00:10:31
tyrosine kinases. So,
after the Epo receptor has
139
00:10:31 --> 00:10:35
bound its liggon, here's
the plasma membrane,
140
00:10:35 --> 00:10:40
it has a whole series of
I'll draw Y here for tyrosine.
141
00:10:40 --> 00:10:43
It has a whole series of phosphates
attached to it because of the
142
00:10:43 --> 00:10:47
actions of tyrosine kinase enzymes
that are associated with its
143
00:10:47 --> 00:10:51
cytoplasmic domain indirect analogy
to what we talked about in the case
144
00:10:51 --> 00:10:54
of growth factor receptors. But,
one of the things that happens
145
00:10:54 --> 00:10:58
is that this phosphotase, which
removes phosphates, then gloms
146
00:10:58 --> 00:11:02
onto the receptor like this.
It grabs hold of some of these
147
00:11:02 --> 00:11:06
tyrosine kinases. And what
this phosphotase does is
148
00:11:06 --> 00:11:10
reach around. It reaches around and
it begins to prune off all of these
149
00:11:10 --> 00:11:14
phosphates because that's
what a phosphate does.
150
00:11:14 --> 00:11:19
It cuts away all the phosphates,
thereby directly reversing the
151
00:11:19 --> 00:11:23
previous actions of the tyrosine
kinase that led to the formation of
152
00:11:23 --> 00:11:27
these phosphates, and that
in turn allows downstream
153
00:11:27 --> 00:11:32
signaling to occur. This
is obviously a functional
154
00:11:32 --> 00:11:36
negative feedback loop, i.e.
whenever there is an agonist
155
00:11:36 --> 00:11:40
you want an antagonist.
Whenever there's a stimulus which
156
00:11:40 --> 00:11:44
is induced in the body, there
has to be an inhibitory signal,
157
00:11:44 --> 00:11:48
and this is part of the
whole issue of homeostasis,
158
00:11:48 --> 00:11:52
the balance between forward and
backward. Interestingly enough,
159
00:11:52 --> 00:11:56
there's a family in Finland, I
believe, which has a mutant receptor.
160
00:11:56 --> 00:12:01
And their mutant receptor
lacks this tyrosine.
161
00:12:01 --> 00:12:04
And what happens as a consequence
is that that particular tyrosine
162
00:12:04 --> 00:12:07
doesn't get phosphorolated.
Because that tyrosine doesn't get
163
00:12:07 --> 00:12:11
phosphorolated, the
phosphotase cannot be attracted
164
00:12:11 --> 00:12:14
to the receptor because
there isn't a tyrosine there.
165
00:12:14 --> 00:12:18
There's some other amino acid
residue. I don't know what it is.
166
00:12:18 --> 00:12:21
It's not important, but it's not
a tyrosine. And this cannot happen
167
00:12:21 --> 00:12:24
because they don't have this
tyrosine. This phosphotase could
168
00:12:24 --> 00:12:28
not be attracted to the receptor
to shut it down as it normally
169
00:12:28 --> 00:12:32
would be. So normally
homeostasis is
170
00:12:32 --> 00:12:36
imbalanced, and several members
of this family have become Olympic
171
00:12:36 --> 00:12:41
cross-country ski winners.
They've become Olympic champions.
172
00:12:41 --> 00:12:45
Why? Because their Epo receptor's
hyperactive. Because the Epo
173
00:12:45 --> 00:12:49
receptor's hyperactive, they
have higher than normal levels
174
00:12:49 --> 00:12:54
of red blood cells in the
circulation, and this clearly allows
175
00:12:54 --> 00:12:58
them to function better
in cross country skiing,
176
00:12:58 --> 00:13:03
which as you know is a really
physically demanding task.
177
00:13:03 --> 00:13:06
Again, I'm not saying this is a
good thing for them necessarily.
178
00:13:06 --> 00:13:10
There are other things in life
besides, believe it or not,
179
00:13:10 --> 00:13:14
winning cross country Olympic
competitions because as I mentioned
180
00:13:14 --> 00:13:18
last time, having too many red
blood cells in your circulation,
181
00:13:18 --> 00:13:22
there's a downside to it which
is that you have a much greater
182
00:13:22 --> 00:13:26
tendency to have occlusions,
to have blood clots in your
183
00:13:26 --> 00:13:30
circulation which obviously is
not a very good thing to have.
184
00:13:30 --> 00:13:38
Oh, so is there
a threshold of Epo
185
00:13:38 --> 00:13:41
receptor activation before
phosphotase shuts it down?
186
00:13:41 --> 00:13:44
These things are not really well
understood, are not well studied.
187
00:13:44 --> 00:13:47
The fact is, you might be able to
say we should make a mathematical
188
00:13:47 --> 00:13:51
model of all of these different
circuitry. But the fact is if you
189
00:13:51 --> 00:13:54
want to make a mathematical
model, you have to know some of the
190
00:13:54 --> 00:13:57
constants. You have to know some
of the parameters, the binding
191
00:13:57 --> 00:14:00
constants. And in
fact, for most of the
192
00:14:00 --> 00:14:04
signaling interactions, no
one's ever really studied them in
193
00:14:04 --> 00:14:08
such great detail. So,
one really doesn't know how
194
00:14:08 --> 00:14:11
much phosphate you need here before
the phosphotase becomes really
195
00:14:11 --> 00:14:15
active. And so, there's
not a really good
196
00:14:15 --> 00:14:19
mathematical model of this feedback
loop, even though we know without
197
00:14:19 --> 00:14:22
any doubt that it exists. So,
I want to get into other issues
198
00:14:22 --> 00:14:26
that are related to the whole
issue of accumulated differentiation
199
00:14:26 --> 00:14:30
traits as one moves
down this pathway.
200
00:14:30 --> 00:14:34
Again, we've used this as a model
for how differentiation takes place
201
00:14:34 --> 00:14:38
in the entire body. The
faith that's been implicit in
202
00:14:38 --> 00:14:42
this kind of scheme for the last 20
or 30 years is that this acquisition
203
00:14:42 --> 00:14:47
of different kinds of phenotypes is
not accompanied by genetic changes,
204
00:14:47 --> 00:14:51
that is, in the genomes of these
cells. I.e. one can accomplish
205
00:14:51 --> 00:14:55
these different kinds of
differentiation not by rearranging
206
00:14:55 --> 00:15:00
genes but just by rearranging
transcriptional programs,
207
00:15:00 --> 00:15:03
and that the DNA sequence of
these cells as they proliferate and
208
00:15:03 --> 00:15:07
differentiate is fully unchanged.
And that's a matter of faith
209
00:15:07 --> 00:15:11
because you could say to me,
how do you know that it's really
210
00:15:11 --> 00:15:15
true. The fact is that people have
looked at genes in many kinds of
211
00:15:15 --> 00:15:18
cell types, but it's essentially
impossible, or it has been at least
212
00:15:18 --> 00:15:22
until recently, to preclude
the possibility that as
213
00:15:22 --> 00:15:26
cells move down these
differentiation pathways,
214
00:15:26 --> 00:15:30
they begin to change the
nucleotide sequences of different
215
00:15:30 --> 00:15:33
ones of their genes. In fact,
I've already told you about
216
00:15:33 --> 00:15:37
one instance where that's clearly
the case. And that is in the
217
00:15:37 --> 00:15:41
differentiation of the B
cells of the immune system,
218
00:15:41 --> 00:15:45
which happen to be right up here
on this chart, because as you recall
219
00:15:45 --> 00:15:48
from our discussion vis-à-vis
immunology, the B cells actually do
220
00:15:48 --> 00:15:52
rearrange their genes in order to
cobble together DNA sequences that
221
00:15:52 --> 00:15:56
together are able to enable them
to make antibodies that are able to
222
00:15:56 --> 00:16:00
react to specific antigens. So
there, there's no doubt at all
223
00:16:00 --> 00:16:04
that there's a somatic rearrangement
of the genes, somatic meaning it's
224
00:16:04 --> 00:16:08
not a germ line change. It's
happening in the soma outside
225
00:16:08 --> 00:16:12
of the germ line. There's
a somatic mutation.
226
00:16:12 --> 00:16:16
It's not a mutation that's
deleterious, but rather is directed
227
00:16:16 --> 00:16:20
towards a physiologically
normal and desirable end point.
228
00:16:20 --> 00:16:24
But for example, how do you know
that when you remember things in the
229
00:16:24 --> 00:16:28
brain, part of the memory does
not derive from changing the DNA
230
00:16:28 --> 00:16:32
sequence and different
neurons in the brain?
231
00:16:32 --> 00:16:36
What's the molecular basis of memory?
Could it be that each time we learn
232
00:16:36 --> 00:16:41
some things that there are
different nucleotide sequences,
233
00:16:41 --> 00:16:45
critical nucleotide sequences,
that are changed in neurons in the
234
00:16:45 --> 00:16:50
brain, and that those nucleotide
sequence changes represent an
235
00:16:50 --> 00:16:54
important basis for ensuring that
memory is retained over decades of
236
00:16:54 --> 00:16:59
time. Or, rather than having
genetic changes in the brain,
237
00:16:59 --> 00:17:03
might it all be epigenetic, i. .
all the other changes that happen
238
00:17:03 --> 00:17:08
to the cell besides changing DNA
sequences in the chromosomal DNA.
239
00:17:08 --> 00:17:13
So, here we're dealing with the
dialectic between epigenetic and
240
00:17:13 --> 00:17:19
genetic. And, have we
talked about DNA methylation
241
00:17:19 --> 00:17:24
here? Yes, so we talked about DNA
methylation, and do you recall or
242
00:17:24 --> 00:17:30
having discussed the fact
that when DNA gets methylated,
243
00:17:30 --> 00:17:36
that suppresses the
transcription of a gene.
244
00:17:36 --> 00:17:39
But that doesn't change
the nucleotide sequence,
245
00:17:39 --> 00:17:43
and that methylation configuration
of a gene can be passed to one cell
246
00:17:43 --> 00:17:46
generation to the next.
It's heritable, but it's not
247
00:17:46 --> 00:17:50
genetic in the strictest
sense of the term, i.e.
248
00:17:50 --> 00:17:54
it doesn't involve a change
in nucleotide sequence,
249
00:17:54 --> 00:17:58
which is what we want to
limit this term to referring.
250
00:17:58 --> 00:18:02
So, epigenic can represent all the
changes in the cell including DNA
251
00:18:02 --> 00:18:07
methylation, alterations
in transcription,
252
00:18:07 --> 00:18:12
and all other downstream events
that result in changes in the cell.
253
00:18:12 --> 00:18:17
And how can one address this?
Well, there are different ways of
254
00:18:17 --> 00:18:22
addressing this question or
addressing the possibility that in
255
00:18:22 --> 00:18:27
fact there are changes in the
nucleotide sequence of the gene.
256
00:18:27 --> 00:18:32
One way to do this is the following.
And that is to take cells from an
257
00:18:32 --> 00:18:37
early embryo, and here we see
an early vertebrate embryo.
258
00:18:37 --> 00:18:42
This looks really more like a frog
embryo or a slightly different shape,
259
00:18:42 --> 00:18:47
and here we see an early embryo.
It's after a blastula. It's called
260
00:18:47 --> 00:18:52
a blastocyst. Here again
we have the word blast.
261
00:18:52 --> 00:18:57
How about one question per lecture?
We have to have some equity here.
262
00:18:57 --> 00:19:02
Other people can ask questions.
It's good to ask questions,
263
00:19:02 --> 00:19:06
but how about one per lecture;
that's fair, equitable.
264
00:19:06 --> 00:19:10
All right, so here's an
early vertebrate embryo.
265
00:19:10 --> 00:19:14
Here we see the blastocyst. This
comes after the earlier stages
266
00:19:14 --> 00:19:18
in the embryo, and here
we see the inner cell mass.
267
00:19:18 --> 00:19:22
And as it turns out, the inner cell
mass is going to be the precursor of
268
00:19:22 --> 00:19:26
many of the tissues of the
ultimately arising embryo.
269
00:19:26 --> 00:19:30
And here, one can do an interesting
experiment. One can take cells out
270
00:19:30 --> 00:19:34
of the inner cell mass. And
one can begin to propagate them
271
00:19:34 --> 00:19:38
in culture. And what one ends
up with is embryonic stem cells.
272
00:19:38 --> 00:19:42
And the intrinsic interest of
embryonic stem cells is manifold.
273
00:19:42 --> 00:19:46
For one thing, you can take
embryonic stem cells and you can
274
00:19:46 --> 00:19:51
genetically alter them.
You can put a new gene in,
275
00:19:51 --> 00:19:55
in the case of a mouse, or
you can take another gene out.
276
00:19:55 --> 00:19:59
And then what you can do is you
can inject the genetically altered
277
00:19:59 --> 00:20:04
embryonic stem cell into the
blastocyst of another embryo.
278
00:20:04 --> 00:20:08
So let's say we take the cells
out of the inner cell mass.
279
00:20:08 --> 00:20:13
We develop embryonic stem cells.
We can call them ES cells. That's
280
00:20:13 --> 00:20:17
what they're called in the trade,
ES cells. We take them out. We can
281
00:20:17 --> 00:20:22
propagate them in culture. And
then, what we can find is we'll
282
00:20:22 --> 00:20:26
put a genetic marker in those ES
cells. Let's say we put in those
283
00:20:26 --> 00:20:31
embryonic stem cells the marker
for the gene beta-galactosidase.
284
00:20:31 --> 00:20:35
And beta-galactosidase in the
presence of a proper indicator,
285
00:20:35 --> 00:20:39
if you put a proper indicator
and make a cell turn blue.
286
00:20:39 --> 00:20:43
So now we have an ES cell line
that produces the beta-galactosidase
287
00:20:43 --> 00:20:47
enzyme. The beta-galactosidase
enzyme beta-gal itself has no effect
288
00:20:47 --> 00:20:51
on the biology of the cells.
It's only a marker. And now,
289
00:20:51 --> 00:20:55
we take those ES cells, and we
inject them into another embryo,
290
00:20:55 --> 00:21:00
a wild type embryo that
lacks this beta-gal marker.
291
00:21:00 --> 00:21:05
And what we can see is that we
inject the ES cells into this
292
00:21:05 --> 00:21:10
blastocyst. The injected ES cells
will now insinuate themselves,
293
00:21:10 --> 00:21:15
will now intrude into the massive
cells in this embryo into which we
294
00:21:15 --> 00:21:20
injected the ES cells, and
they will become part of the
295
00:21:20 --> 00:21:25
entire embryo genesis that follows.
I.e. soon these foreign ES cells
296
00:21:25 --> 00:21:30
will weasel their way
into this inner cell mass.
297
00:21:30 --> 00:21:34
And they will become established
and become functionally equivalent to
298
00:21:34 --> 00:21:38
the inner cell mass cells that
were resident there prior to this
299
00:21:38 --> 00:21:42
injection. And what you can do then
is follow the subsequent fate of,
300
00:21:42 --> 00:21:46
in this case, a mouse. And what
will happen often is that you can
301
00:21:46 --> 00:21:50
find blue cells all over the
mouse sometimes in the paws,
302
00:21:50 --> 00:21:54
sometimes in the coat. Let's
imagine that the hair would turn
303
00:21:54 --> 00:21:58
blue, which in fact is not the case.
But let's imagine the hair would
304
00:21:58 --> 00:22:02
turn blue. So
here's the mouse,
305
00:22:02 --> 00:22:06
happy because it's part
of an important experiment.
306
00:22:06 --> 00:22:11
And what you'll sometimes see is
that, well, remember that art was
307
00:22:11 --> 00:22:16
not my forte. Anyhow, here
you might see stripes of blue
308
00:22:16 --> 00:22:20
cells on the skin. The hair
won't turn blue actually,
309
00:22:20 --> 00:22:25
but the skin may if you
give it the proper indicator.
310
00:22:25 --> 00:22:29
And what this indicates is that
in this case, the cells that were
311
00:22:29 --> 00:22:34
injected into the blastocyst
could become part of lineages which
312
00:22:34 --> 00:22:39
committed themselves
to becoming skin cells.
313
00:22:39 --> 00:22:43
Or, the cells in the brain might
be blue. Or, the cells in the gut
314
00:22:43 --> 00:22:47
might be blue. Or under
certain conditions,
315
00:22:47 --> 00:22:51
the cells in the intestine might
be blue. In telling you that,
316
00:22:51 --> 00:22:55
I mean to indicate that the
cells that we injected into this
317
00:22:55 --> 00:23:00
blastocyst, which carry
beta-gal were totipotent.
318
00:23:00 --> 00:23:04
They could create all the tissues
of the mouse under the proper
319
00:23:04 --> 00:23:08
conditions. The proper conditions
are obviously being put into this
320
00:23:08 --> 00:23:12
very special environment in
which all kinds of differentiation
321
00:23:12 --> 00:23:16
inducing signals, which
we don't really understand,
322
00:23:16 --> 00:23:20
can induce this cell to commit
itself to enter into one or another
323
00:23:20 --> 00:23:24
differentiation lineage. And
in principal, you can make a
324
00:23:24 --> 00:23:28
whole organism out of an ES cell.
ES cell has as much plasticity, as
325
00:23:28 --> 00:23:32
much flexibility,
as a fertilized egg.
326
00:23:32 --> 00:23:36
It has not yet lost the ability
to make all the parts of the body.
327
00:23:36 --> 00:23:40
On some occasions, the ES cell
will even get into the gonads of the
328
00:23:40 --> 00:23:45
mouse, which are down here
somewhere. And if that's so,
329
00:23:45 --> 00:23:49
if the ES cell which you injected
has been able to seed the formation
330
00:23:49 --> 00:23:54
of these cells down here, then
what will happen is that either
331
00:23:54 --> 00:23:58
the sperm or the egg coming
from this mouse will now transmit
332
00:23:58 --> 00:24:04
the blue gene. And now,
in the next generation,
333
00:24:04 --> 00:24:10
all of the mice will inherit the
blue beta-galactosidase gene in all
334
00:24:10 --> 00:24:16
of their cells because now this
will have entered into the germ line.
335
00:24:16 --> 00:24:22
If these blue cells happen
to colonize the testes,
336
00:24:22 --> 00:24:28
the ovary, or the testes, then
these blue cells will become
337
00:24:28 --> 00:24:32
ancestors to the sperm or the egg.
And now, in the next generation,
338
00:24:32 --> 00:24:36
mice will inherit a blue
gene in all of their cells.
339
00:24:36 --> 00:24:40
And now this mouse is really
happy because it's now part of an
340
00:24:40 --> 00:24:44
extremely important experiment
because now all of its cells will
341
00:24:44 --> 00:24:47
become blue, having inherited them
as part of the oocyte which led to
342
00:24:47 --> 00:24:51
its formation. In
this kind of an animal,
343
00:24:51 --> 00:24:55
we call this animal a kind of
a chimera. Chimera is a mythical
344
00:24:55 --> 00:24:59
beast which is, let's say,
half human and half horse
345
00:24:59 --> 00:25:02
or something like that.
Or a chimera means it has
346
00:25:02 --> 00:25:06
genetically different parts in it.
That is not to say that these parts
347
00:25:06 --> 00:25:09
carrying the blue gene
are necessarily defective,
348
00:25:09 --> 00:25:13
they're just genetically different,
one from the other. But they can
349
00:25:13 --> 00:25:16
participate in embryogenesis in
a fashion that's indistinguishable
350
00:25:16 --> 00:25:20
from the non-blue cells. They
just do everything they're
351
00:25:20 --> 00:25:23
supposed to do, and they
pretend as if they were in
352
00:25:23 --> 00:25:27
this embryo from the get go,
from the very beginning, from the
353
00:25:27 --> 00:25:31
moment of fertilization.
So they are totipotent.
354
00:25:31 --> 00:25:34
There's an alternative experiment
you can do, and you can take the ES
355
00:25:34 --> 00:25:38
cells, and you can inject
them under the skin of a mouse,
356
00:25:38 --> 00:25:41
let's say. So now, you're
putting them in a very unfamiliar
357
00:25:41 --> 00:25:45
environment. And what you see
then on many occasions is you can
358
00:25:45 --> 00:25:49
actually get a tumor. You
can get what's called an
359
00:25:49 --> 00:26:00
embryonal carcinoma.
360
00:26:00 --> 00:26:03
Now you'll say, well,
so what? That's not so
361
00:26:03 --> 00:26:07
interesting. But it's
very interesting. Why?
362
00:26:07 --> 00:26:10
Because if you look at the genome
of those embryonal carcinoma cells
363
00:26:10 --> 00:26:14
which we can call EC cells if you
want, those cells are genetically
364
00:26:14 --> 00:26:17
full wild type. And yet,
we're getting a tumor here.
365
00:26:17 --> 00:26:21
So, it means that these cells,
which have been placed in a fully
366
00:26:21 --> 00:26:24
unfamiliar environment under the
skin or in the belly of a mouse will
367
00:26:24 --> 00:26:28
begin to form a tumor. And
in fact, they represent the
368
00:26:28 --> 00:26:31
only type of cell that we know
about where a cell having a wild type
369
00:26:31 --> 00:26:35
genome can actually
give you a tumor.
370
00:26:35 --> 00:26:39
As you sensed from our previous
discussions, all other kinds of
371
00:26:39 --> 00:26:44
human cancer cells we know about
have to have mutant genes in order
372
00:26:44 --> 00:26:48
for them to grow as a malignancy.
These cells are fully wild type and
373
00:26:48 --> 00:26:53
can grow as an embryonal carcinoma.
They are very primitive. These
374
00:26:53 --> 00:26:57
cells have quite a bit of autonomy.
They're not so responsive to all
375
00:26:57 --> 00:27:02
the growth factors that normally
are required by many cells throughout
376
00:27:02 --> 00:27:07
the soma of an animal
throughout the tissues.
377
00:27:07 --> 00:27:10
So this allows us to begin to move
on and ask other kinds of questions.
378
00:27:10 --> 00:27:14
For example, you can take
these embryonal carcinoma cells.
379
00:27:14 --> 00:27:18
You put them in a Petri dish, and
you can actually induce them to
380
00:27:18 --> 00:27:22
differentiate into different
cell types in vitro.
381
00:27:22 --> 00:27:26
How can you do that? Well,
we're just beginning to learn
382
00:27:26 --> 00:27:30
how to do that. We don't
really know how to do that.
383
00:27:30 --> 00:27:34
But, if you give them the right
cocktail of growth factors,
384
00:27:34 --> 00:27:38
they might begin to form muscle
cells. If you give them another
385
00:27:38 --> 00:27:43
cocktail of growth factors, they
might begin to give pancreatic
386
00:27:43 --> 00:27:47
eyelid cells that form insulin,
or in this case cartilage cells.
387
00:27:47 --> 00:27:52
And presumably, the cocktail of
growth factors you're providing each
388
00:27:52 --> 00:27:56
one of these cells with in vitro,
i.e. in the Petri dish, is mimicking
389
00:27:56 --> 00:28:00
the growth factor environment
that each of these cell types is
390
00:28:00 --> 00:28:04
experiencing within the
embryo. In other words,
391
00:28:04 --> 00:28:08
cells in different parts of
the embryo experience different
392
00:28:08 --> 00:28:12
combinations of growth factors that
persuade them to commit themselves
393
00:28:12 --> 00:28:16
to becoming these kind of cells,
these kind of cells, and these kind
394
00:28:16 --> 00:28:20
of cells. And therefore, one
of the promises of embryonic
395
00:28:20 --> 00:28:24
stem cell research is the
possibility of being able to
396
00:28:24 --> 00:28:28
regenerate different kinds of
tissues in a fashion that I just
397
00:28:28 --> 00:28:32
showed you here. But this
whole experiment in the
398
00:28:32 --> 00:28:36
case of human beings is
ethically extremely controversial.
399
00:28:36 --> 00:28:40
Why? Because the experiment starts
out making these ES cells here,
400
00:28:40 --> 00:28:44
and if we want to start out
with an early embryo like this,
401
00:28:44 --> 00:28:48
start out with a blastocyst, in
the case of a human blastocyst,
402
00:28:48 --> 00:28:52
this human blastocyst has
the potential under the proper
403
00:28:52 --> 00:28:56
conditions of becoming a newborn
human being. And therefore,
404
00:28:56 --> 00:29:00
we have this enormous ethical
conflict in this country.
405
00:29:00 --> 00:29:04
Is this blastocyst already a human
being? Can you already afford to
406
00:29:04 --> 00:29:08
truncate the life of this blastocyst
at this stage of development,
407
00:29:08 --> 00:29:13
and in so doing, are you
actually extinguishing human life,
408
00:29:13 --> 00:29:17
or is this organism, if you want to
call it that, already still much too
409
00:29:17 --> 00:29:22
primitive to consider it
to be equal to human life?
410
00:29:22 --> 00:29:26
And here, I would not,
unlike my political views,
411
00:29:26 --> 00:29:31
be forward enough to venture
an opinion because it's really
412
00:29:31 --> 00:29:35
something that no one really can
argue about in any objective way.
413
00:29:35 --> 00:29:40
It's all a matter of opinion.
Is this a human being already,
414
00:29:40 --> 00:29:44
or is it simply an inanimate
cluster, a clump of cells?
415
00:29:44 --> 00:29:48
Now, in principal,
how could we do this?
416
00:29:48 --> 00:29:52
How could we actually create
this kind of tissue therapy?
417
00:29:52 --> 00:29:56
Because the fact is, as you get
older, your tissues start falling
418
00:29:56 --> 00:30:00
apart. You haven't
experienced that.
419
00:30:00 --> 00:30:04
But I have. And the fact is that
even if you try to stay in shape,
420
00:30:04 --> 00:30:09
things just start falling
apart. And the older you get,
421
00:30:09 --> 00:30:13
the more they fall apart.
Even people who eat well,
422
00:30:13 --> 00:30:18
which I do, and exercise well,
which I don't, even they fall apart.
423
00:30:18 --> 00:30:22
And so the question is, are there
way of replacing and repairing
424
00:30:22 --> 00:30:27
tissue? And this would, in
principal, represent one such
425
00:30:27 --> 00:30:31
strategy because it means that you
could possibly inject replacement
426
00:30:31 --> 00:30:36
cells into an agent tissue and
generate cells which could then
427
00:30:36 --> 00:30:40
restore and regeneration function
which has somehow inevitably
428
00:30:40 --> 00:30:45
deteriorated over the decades.
Well, that raises the question of
429
00:30:45 --> 00:30:50
how you can actually get a
blastocyst, how you can make a
430
00:30:50 --> 00:30:56
blastocyst like this. To state
an obvious thing which you
431
00:30:56 --> 00:31:01
might already have intuited,
let's say you had such cells
432
00:31:01 --> 00:31:05
differentiated from various cell
types that you want to inject into
433
00:31:05 --> 00:31:09
somebody's muscle or into their
liver if they had diabetes and had
434
00:31:09 --> 00:31:13
lost their beta cells, or
into their cartilage if they
435
00:31:13 --> 00:31:17
banged up their knee during
basketball practice or something
436
00:31:17 --> 00:31:21
like that, or jogging, which
is allegedly good for you.
437
00:31:21 --> 00:31:25
Who knows? How could you deal
with that? Well, the fact is,
438
00:31:25 --> 00:31:29
let's imagine there were such a
blastocyst which we'd produce in
439
00:31:29 --> 00:31:34
this fashion that we
differentiated like this.
440
00:31:34 --> 00:31:37
OK, this is now the sequence
of events. There's an important
441
00:31:37 --> 00:31:40
consideration we have to take
into account, and that is if this
442
00:31:40 --> 00:31:44
blastocyst came from a
different person than you,
443
00:31:44 --> 00:31:47
and we induced these
cells to differentiate,
444
00:31:47 --> 00:31:51
and we injected those
differentiation cells into your
445
00:31:51 --> 00:31:54
muscle, things wouldn't work.
Why? Because these cells, if the
446
00:31:54 --> 00:31:57
blastocyst originated in a different
person than yourself would be
447
00:31:57 --> 00:32:01
genetically different from you,
and would be recognized as foreign
448
00:32:01 --> 00:32:04
tissue by your immune system. So
even though you were getting an
449
00:32:04 --> 00:32:08
injection of cells which could
regenerate your muscle perfectly
450
00:32:08 --> 00:32:11
well, those cells would never
be given a chance to establish
451
00:32:11 --> 00:32:15
themselves and to thrive, and
to reconstruct the tissue simple
452
00:32:15 --> 00:32:18
because the immune system would
regard those cells as being
453
00:32:18 --> 00:32:22
foreigners and would go after them
hammer and tongs trying to get rid
454
00:32:22 --> 00:32:25
of them in the same way it tries
to get rid of all kinds of foreign
455
00:32:25 --> 00:32:29
invaders. I.e. the only
way you could avoid it is
456
00:32:29 --> 00:32:33
if this blastocyst was
genetically identical to you.
457
00:32:33 --> 00:32:37
But how can you make a blastocyst
which is genetically identical to
458
00:32:37 --> 00:32:41
you? Well, I'm glad I asked that
question. That's really the big
459
00:32:41 --> 00:32:45
challenge we have here because we
don't want to create a situation
460
00:32:45 --> 00:32:49
where we have to restore somebody's
tissues, but the only way we can
461
00:32:49 --> 00:32:53
restore them is to leave them
immunosuppressed for the rest of
462
00:32:53 --> 00:32:57
their lives. When I say
immunosuppressed I mean we have to
463
00:32:57 --> 00:33:01
prevent their immune system from
attacking all of these cells that
464
00:33:01 --> 00:33:05
we've injected in them, these
foreign cells, in the same way
465
00:33:05 --> 00:33:09
that we have to suppress the
immune system of any person who has
466
00:33:09 --> 00:33:13
received a graft from another
individual including often bone
467
00:33:13 --> 00:33:18
marrow transplants. In all
cases, we have at least for a
468
00:33:18 --> 00:33:24
while to prevent their immune system
from attacking and eliminating these
469
00:33:24 --> 00:33:29
engrafted cells. And this
is where the whole strategy
470
00:33:29 --> 00:33:33
comes for the whole process of
cloning. You may recall the case of
471
00:33:33 --> 00:33:37
Dolly about five years ago,
and let's remember what happened
472
00:33:37 --> 00:33:41
here because this would a momentous
experiment in mammalian biology.
473
00:33:41 --> 00:33:45
It asked the question, really, if
you take cells from a somatic tissue,
474
00:33:45 --> 00:33:49
from here, or here, or
here, are those cells,
475
00:33:49 --> 00:33:53
in principal, still totipotent,
i.e. is the nucleus, is the genome
476
00:33:53 --> 00:33:57
of those cells totipotent, or
has the genome, the chromosomal
477
00:33:57 --> 00:34:01
complement of cells in their cells
undergone some kind of irrevocable,
478
00:34:01 --> 00:34:05
irreversible change, which
precludes those cells from ever
479
00:34:05 --> 00:34:08
becoming totipotent?
Well, in fact,
480
00:34:08 --> 00:34:12
if you take mammary epithelial cells
from the breast of a human being or
481
00:34:12 --> 00:34:15
from the breast of a ewe and
you put them into the blastocyst,
482
00:34:15 --> 00:34:18
nothing's going to happen. Those
introduced mammary epithelial
483
00:34:18 --> 00:34:22
cells will not be able to establish
themselves in the blastocyst.
484
00:34:22 --> 00:34:25
And, we will not be able to
insinuate themselves amidst the
485
00:34:25 --> 00:34:29
inner cell mass, and
they will not be able to
486
00:34:29 --> 00:34:33
participate in embryogenesis. So
therefore, the epigenetic program
487
00:34:33 --> 00:34:38
in these somatic cells seems to
be irrevocably set to preclude the
488
00:34:38 --> 00:34:44
participation of the already
differentiated mammary epithelial
489
00:34:44 --> 00:34:49
cells in subsequent embryogenesis.
Therefore, you could not do this
490
00:34:49 --> 00:34:54
experiment all over again of
introducing cells into the inner
491
00:34:54 --> 00:35:00
cell mass as I just described over
here, injecting them into this.
492
00:35:00 --> 00:35:04
But still, that doesn't answer the
question. The issue is not whether
493
00:35:04 --> 00:35:08
the mammary epithelial cell is
irrevocably committed to being a
494
00:35:08 --> 00:35:12
mammary epithelial cell. The
issue: is its genome capable
495
00:35:12 --> 00:35:16
under the proper circumstances of
becoming an early embryonic cell.
496
00:35:16 --> 00:35:21
And therefore, what was done is
the following. One took mammary
497
00:35:21 --> 00:35:25
epithelial cells, in this
case from Dolly's quote
498
00:35:25 --> 00:35:29
unquote "mother, one
prepared nuclei from these
499
00:35:29 --> 00:35:33
cells, taking them out of the
cytoplasm, and then one got
500
00:35:33 --> 00:35:38
fertilized eggs or eggs that
have been induced to become.
501
00:35:38 --> 00:35:42
So here's an oocyte. An
oocyte is an unfertilized egg.
502
00:35:42 --> 00:35:46
In principle, you can activate
an oocyte by putting a sperm in,
503
00:35:46 --> 00:35:51
or in fact it's actually better if
you take the oocyte and you fool it
504
00:35:51 --> 00:35:55
into thinking it's become fertilized
by treating it with different salts,
505
00:35:55 --> 00:36:00
high potassium
concentration, and so forth.
506
00:36:00 --> 00:36:04
And that will induce the egg
to say I've been fertilized.
507
00:36:04 --> 00:36:09
I better start embryogenesis. But
what you do in this case is the
508
00:36:09 --> 00:36:13
following. The egg has its
own haploid nucleus here,
509
00:36:13 --> 00:36:18
and you can take a little needle.
And, you suck that nucleus right
510
00:36:18 --> 00:36:23
out of the egg. So,
you've enucleated it.
511
00:36:23 --> 00:36:27
That's what you've done,
and now the egg is enucleate.
512
00:36:27 --> 00:36:32
It doesn't have a nucleus
in it. But keep in mind,
513
00:36:32 --> 00:36:36
much of what happens during early
embryogenesis is programmed not only
514
00:36:36 --> 00:36:41
by the genes but by all array
of cytoplasmic proteins that are
515
00:36:41 --> 00:36:46
present throughout the egg,
and which play critical roles in
516
00:36:46 --> 00:36:50
determining the subsequent
course of embryogenesis.
517
00:36:50 --> 00:36:55
So now what you can do is you
inject into this enucleate oocyte
518
00:36:55 --> 00:37:00
the nucleus of a
mammary epithelial cell.
519
00:37:00 --> 00:37:05
The mammary epithelial cell is
obviously highly differentiated.
520
00:37:05 --> 00:37:10
It's there to make milk. We'll
call it an MEC if you want,
521
00:37:10 --> 00:37:15
and you put that in there, and
under certain circumstances,
522
00:37:15 --> 00:37:20
and then you can treat this with
a little bit of salt to mimic the
523
00:37:20 --> 00:37:25
physiological stimulus that comes
after the sperm hits the egg.
524
00:37:25 --> 00:37:31
And now this egg will
think it's been fertilized.
525
00:37:31 --> 00:37:35
And now it will begin to divide.
But keep in mind, the genome of
526
00:37:35 --> 00:37:39
this quote unquote "unfertilized
egg" has come not from the sperm and
527
00:37:39 --> 00:37:44
the preexisting nucleus of the egg.
It's come because the nucleus has
528
00:37:44 --> 00:37:48
been injected from a
mammary epithelial cell.
529
00:37:48 --> 00:37:52
An experience over the last 30
years had indicated that this will
530
00:37:52 --> 00:37:57
never work. But finally somebody in
Scotland, a man named Ian Wilmouth
531
00:37:57 --> 00:38:01
tinkered enough with the conditions
of these cells that he could
532
00:38:01 --> 00:38:05
actually get it to work not
so often, maybe one, or two,
533
00:38:05 --> 00:38:10
or three times out of 100
tries. But on those conditions,
534
00:38:10 --> 00:38:14
this thing would begin to divide.
The nucleus would begin to divide
535
00:38:14 --> 00:38:19
its diploid. Keep in mind that
when a sperm comes into an egg,
536
00:38:19 --> 00:38:23
the egg is haploid. The sperm
is haploid. Together they make a
537
00:38:23 --> 00:38:27
diploid genome. This
introduced genomus diploid,
538
00:38:27 --> 00:38:32
and the question is, the critical
question is, can the genes in this
539
00:38:32 --> 00:38:36
introduced nucleus totally rearrange
their transcriptional program so
540
00:38:36 --> 00:38:41
that even though these genes might
all be intact in terms of nucleotide
541
00:38:41 --> 00:38:45
sequence, can the entire infinitely
complex array of DNA associated
542
00:38:45 --> 00:38:50
proteins, I.e. the
proteins that constitute
543
00:38:50 --> 00:38:54
the chromatin which is not only the
histones but also the transcription
544
00:38:54 --> 00:38:59
factors, the TF's, can they
all jump on and jump off as
545
00:38:59 --> 00:39:03
they should to mimic and replicate
the spectrum of transcription
546
00:39:03 --> 00:39:08
factors that is normally present
shortly after an egg is fertilized?
547
00:39:08 --> 00:39:12
If they can do that, then
this embryo can begin to
548
00:39:12 --> 00:39:16
replicate, and can ultimately
develop into a complete embryo.
549
00:39:16 --> 00:39:20
If they can't, then embryogenesis
is going to be truncated shortly
550
00:39:20 --> 00:39:24
thereafter maybe at the two cell
stage, at the four cell stage,
551
00:39:24 --> 00:39:28
at the 16 cell stage, but shortly
thereafter, not because of the DNA
552
00:39:28 --> 00:39:32
sequences being defective,
but because the spectrum of
553
00:39:32 --> 00:39:36
transcription factors is up and down
regulates certain genes is in fact
554
00:39:36 --> 00:39:40
not been able to re-assort
themselves in response to what?
555
00:39:40 --> 00:39:44
Initially, in response to the
signals coming from the cytoplasm
556
00:39:44 --> 00:39:48
because one might imagine,
correctly so, that the nucleus in
557
00:39:48 --> 00:39:53
here is getting signals from
the cytoplasm telling it,
558
00:39:53 --> 00:39:57
in effect, telling this nucleus,
you should behave functionally as if
559
00:39:57 --> 00:40:01
you were the nucleus of a
fertilized egg. In other words,
560
00:40:01 --> 00:40:05
the environment of proteins
here is influencing the behavior
561
00:40:05 --> 00:40:09
of this nucleus. That goes
backwards to our normal
562
00:40:09 --> 00:40:12
way of thinking because keep in mind
our normal vectoral way of thinking
563
00:40:12 --> 00:40:14
is that the nucleus is
influencing the cytoplasm.
564
00:40:14 --> 00:40:17
That's the direction of
information flow. But here,
565
00:40:17 --> 00:40:20
we're having a different situation.
Here, the cytoplasm is telling this
566
00:40:20 --> 00:40:23
injected nucleus, well,
you used to be a mammary
567
00:40:23 --> 00:40:25
epithelial cell nucleus, but
now you've got to take on a
568
00:40:25 --> 00:40:28
different job. And we're
going to force you to do
569
00:40:28 --> 00:40:32
so. And to the
extent that happens,
570
00:40:32 --> 00:40:36
then in principle, one can
end up having a normal embryo.
571
00:40:36 --> 00:40:40
And, it happened actually on
rare occasion that this worked.
572
00:40:40 --> 00:40:44
Here they used actual electrical
stimulus rather than salt to get the
573
00:40:44 --> 00:40:48
nucleus to divide. This
electrical stimulus,
574
00:40:48 --> 00:40:52
again, was to mimic the stimulus
that the sperm entering the egg
575
00:40:52 --> 00:40:56
normally provides, thereby
activating the egg and
576
00:40:56 --> 00:41:00
forcing the entire
fertilized egg to proliferate.
577
00:41:00 --> 00:41:03
And so, once this starts developing,
let's say, the blastocyst stage,
578
00:41:03 --> 00:41:07
here we have a blastocyst.
You can see the inner cell mass
579
00:41:07 --> 00:41:11
once again here. This
can be transferred into a
580
00:41:11 --> 00:41:14
pseudo-pregnant ewe.
Pseudo-pregnant means you take a
581
00:41:14 --> 00:41:18
female ewe and you inject it with a
series of hormones that persuade her
582
00:41:18 --> 00:41:22
reproductive system including
prolactin, and progesterone,
583
00:41:22 --> 00:41:25
or estrogen, persuade
her reproductive system,
584
00:41:25 --> 00:41:29
her uterus, that she's pregnant.
You inject this early embryo into
585
00:41:29 --> 00:41:33
her, and this early embryo will then
implant into the wall of her uterus
586
00:41:33 --> 00:41:37
and begin to develop.
And if it all works well,
587
00:41:37 --> 00:41:41
you get a Dolly is born. You get
a new sheep coming out of this.
588
00:41:41 --> 00:41:46
It doesn't work so often, one,
two, three, four times after out of
589
00:41:46 --> 00:41:50
a hundred, and very often in
the great majority of cases,
590
00:41:50 --> 00:41:55
there are mis-births, mis-carriages,
which happen in the middle of
591
00:41:55 --> 00:41:59
embryogenesis. So, almost
in the great majority of
592
00:41:59 --> 00:42:04
cases, this fails. Somehow,
the reprogramming of this
593
00:42:04 --> 00:42:08
nucleus, which is what we're talking
about, reprogramming it in terms of
594
00:42:08 --> 00:42:12
its transcriptional program,
goes awry. And therefore, bad
595
00:42:12 --> 00:42:17
things happen. The fact
that on a rare occasion
596
00:42:17 --> 00:42:21
gets and succeeds here already is
extremely interesting because it
597
00:42:21 --> 00:42:25
proves irrevocably that the genome
of a mammary epithelial cell is in
598
00:42:25 --> 00:42:30
principle competent to program
entire embryonic development.
599
00:42:30 --> 00:42:34
And that means that during the
development of Dolly's mother,
600
00:42:34 --> 00:42:39
we'll put her up here, as she
developed from one cell into 1,
601
00:42:39 --> 00:42:44
00 or 10,000 billion cells, as
that development occurred the DNA
602
00:42:44 --> 00:42:49
sequences that went from the
fertilized egg to her didn't really
603
00:42:49 --> 00:42:53
change. I.e. the DNA sequences
that were in one of her mammary
604
00:42:53 --> 00:42:58
epithelial cells were intact,
and as capable in principle of
605
00:42:58 --> 00:43:03
launching the full-fledged
development as would be
606
00:43:03 --> 00:43:08
a fertilized egg. And
that is one of the proofs,
607
00:43:08 --> 00:43:12
by the way, that in fact
differentiation does not involve,
608
00:43:12 --> 00:43:16
with some rare exceptions,
alterations in DNA sequence.
609
00:43:16 --> 00:43:20
This, in turn, ends up being
connected with the whole issue of
610
00:43:20 --> 00:43:24
embryonic stem cells. Let's
say that I wanted to have my
611
00:43:24 --> 00:43:28
muscles regenerated, although
they're still pretty good.
612
00:43:28 --> 00:43:33
So, I take a skin cell of mine,
and I inject the skin cell.
613
00:43:33 --> 00:43:36
I take the nucleus out, and
I inject it into an oocyte.
614
00:43:36 --> 00:43:40
And then I let the oocyte
develop up to this stage.
615
00:43:40 --> 00:43:44
And I don't put the oocyte back
into a sheep or another woman,
616
00:43:44 --> 00:43:48
although I could in principle. I
actually take the cells out of the
617
00:43:48 --> 00:43:51
inner cell mass.
Those are ES cells,
618
00:43:51 --> 00:43:55
and I begin to use them to
regenerate my muscles to do this
619
00:43:55 --> 00:43:59
strategy. So, the
cells are, in this case,
620
00:43:59 --> 00:44:03
not used for reproductive cloning,
which is what this is here.
621
00:44:03 --> 00:44:07
They're used for therapeutic cloning,
where instead of taking these cells
622
00:44:07 --> 00:44:11
and the ES cells and allowing
them to form a whole embryo,
623
00:44:11 --> 00:44:15
they're used to form a cell line
of ES cells from the blastocyst from
624
00:44:15 --> 00:44:19
the inner cell mass. What
we talked about before,
625
00:44:19 --> 00:44:23
here you see the blastocyst
with the inner cell mass here.
626
00:44:23 --> 00:44:27
You see it again. But now, rather
than allowing this blastocyst
627
00:44:27 --> 00:44:31
to continue development, we
simply extract cells from it and
628
00:44:31 --> 00:44:34
again create ES cells. I
could create therefore in
629
00:44:34 --> 00:44:38
principle, ES cells, which
are genetically identical to
630
00:44:38 --> 00:44:41
all the cells in my body, and
any one of you could as well.
631
00:44:41 --> 00:44:44
And here, there's not only one, but
there's two ethical complications.
632
00:44:44 --> 00:44:48
First of all, here we're starting
human life with the intent of
633
00:44:48 --> 00:44:51
truncating it very early, and
secondly, where are the oocytes
634
00:44:51 --> 00:44:54
going to come from? Well,
you could say you can get
635
00:44:54 --> 00:44:58
them from some women, but
producing oocytes from a human
636
00:44:58 --> 00:45:02
female isn't so easy. You
have to inject her with all
637
00:45:02 --> 00:45:06
kinds of stimulatory hormones,
choreogramatatrophic hormones. It's
638
00:45:06 --> 00:45:10
an unpleasant procedure.
Usually women are paid $5,
639
00:45:10 --> 00:45:14
00 or $10,000 to produce
some oocytes. Well,
640
00:45:14 --> 00:45:18
you say, that's OK, but
is that OK? I don't know.
641
00:45:18 --> 00:45:22
Is it OK to pay a woman to donate
her oocytes to make herself into an
642
00:45:22 --> 00:45:26
oocyte factory? I don't
know. You have to judge.
643
00:45:26 --> 00:45:30
I think there's arguments
both for and against it.
644
00:45:30 --> 00:45:34
Clearly, any one of us would be
extraordinarily naïve if we thought
645
00:45:34 --> 00:45:39
that this was a procedure which
had no ethical encumbrances in it.
646
00:45:39 --> 00:45:43
And, you have to think about
them for yourself. Still,
647
00:45:43 --> 00:45:48
the potentials are enormous, and
therefore the question exists.
648
00:45:48 --> 00:45:53
Will there be ways in the future
of taking differentiated cells from
649
00:45:53 --> 00:45:57
one's tissue, and in fact using
them in these ways to make ES cells
650
00:45:57 --> 00:46:02
without having to go through an
oocyte, and without having the
651
00:46:02 --> 00:46:06
potential of creating human life.
The alternative to this has been to
652
00:46:06 --> 00:46:10
do the following, to go
into our normal tissues and
653
00:46:10 --> 00:46:14
pull out adult stem cells. What
do I mean by adult stem cells?
654
00:46:14 --> 00:46:18
These are not stem cells that are
totipotent. These are stem cells
655
00:46:18 --> 00:46:22
which are in my muscles and
regenerating muscle mass,
656
00:46:22 --> 00:46:26
which happens believe it or not.
These are stem cells which might be
657
00:46:26 --> 00:46:30
in my skin and are continually
regenerating skin cells.
658
00:46:30 --> 00:46:34
Keep in mind that in the maintenance
of all our normal tissues there are
659
00:46:34 --> 00:46:38
stem cells whose configuration can
formally be depicted like this with
660
00:46:38 --> 00:46:42
the transit amplifying
cells we talked about before.
661
00:46:42 --> 00:46:46
And maybe, if one took the stem
cells out of an adult tissue right
662
00:46:46 --> 00:46:50
here, if we had a way of extracting
them, those could be propagated in
663
00:46:50 --> 00:46:54
vitro, and then injected back
in. Those are so-called adult stem
664
00:46:54 --> 00:46:58
cells. And the
individuals who are against
665
00:46:58 --> 00:47:02
this kind of manipulation of human
embryos and so forth say that adult
666
00:47:02 --> 00:47:06
stem cells are really the solution.
You take stem cells out of a
667
00:47:06 --> 00:47:10
person's tissue, you expand
them. Ex vivo means out
668
00:47:10 --> 00:47:14
of the body, in vitro, and
then you use them. You inject
669
00:47:14 --> 00:47:19
them into somebody's tissue
to regenerate their tissue.
670
00:47:19 --> 00:47:23
There's only one problem with that.
It's ethically far less encumbered
671
00:47:23 --> 00:47:27
obviously, but it doesn't
work that well. In fact,
672
00:47:27 --> 00:47:31
some people think it hardly works at
all, that the exceptions are really
673
00:47:31 --> 00:47:36
rather far and few between. And
so, this issue will long be or
674
00:47:36 --> 00:47:41
continue to be debated. But
it obviously represents a very
675
00:47:41 --> 00:47:46
new and exciting area of biomedical
research. And interestingly enough,
676
00:47:46 --> 00:47:52
it impinges as well in a fully
unexpected way on cancer because
677
00:47:52 --> 00:47:57
this whole paradigm of stem cells,
it turns out, also applies to cancer
678
00:47:57 --> 00:48:01
cells. If you were to
have asked me two or
679
00:48:01 --> 00:48:05
three years ago, what did
the cells in the tumor look
680
00:48:05 --> 00:48:09
like? I would draw a picture like
this, that these are a series of
681
00:48:09 --> 00:48:12
exponentially growing cells
so that all the cancer cells,
682
00:48:12 --> 00:48:16
all the neoplastic cells in
the tumor mass are biologically
683
00:48:16 --> 00:48:20
equivalent to one another.
They all have the same mutant
684
00:48:20 --> 00:48:23
genome, and they all are capable
of multiplying exponentially.
685
00:48:23 --> 00:48:27
But it turns out that work in the
Matavoidic system on Matevoidic
686
00:48:27 --> 00:48:31
tumors like leukemias,
and now on breast cancers,
687
00:48:31 --> 00:48:36
yields a very different results,
because it turns out that the way
688
00:48:36 --> 00:48:44
that the tumors are organized
looks like this. The tumors also are
689
00:48:44 --> 00:48:52
organized in this hierarchical
array just like normal tissue.
690
00:48:52 --> 00:49:00
How do we know that? Again,
I'm glad I asked that question.
691
00:49:00 --> 00:49:04
Because if you take these cells out
of the tumor and put them in another
692
00:49:04 --> 00:49:08
mouse, let's say,
you get a new tumor.
693
00:49:08 --> 00:49:13
These cells are tumorogenic,
I.e. they concede a new tumor.
694
00:49:13 --> 00:49:17
If you take these cells out of the
tumor, they have the same mutant
695
00:49:17 --> 00:49:21
genome. They constitute the bulk,
the vast mass of the cancer cells in
696
00:49:21 --> 00:49:26
a tumor. You put these into a
mouse, and they're non-tumorogenic.
697
00:49:26 --> 00:49:30
And, in some kinds of tumors, the
tumorogenic cells can represent
698
00:49:30 --> 00:49:35
only 1 or 2% of the total mass
of cancer cells in the tumor.
699
00:49:35 --> 00:49:38
And from this, we begin
to realize that you look
700
00:49:38 --> 00:49:42
inside tumors: the tumors deviate
minimally from the organization of
701
00:49:42 --> 00:49:46
normal tissue. They also
depend on self-renewing
702
00:49:46 --> 00:49:50
stem cells which can make transit
amplifying cells and can give end
703
00:49:50 --> 00:49:53
stage cells, which although
they're neoplastic, have many of the
704
00:49:53 --> 00:49:57
differentiated characteristics of
the normal tissue from which they
705
00:49:57 --> 00:50:01
arose. And this has
enormous implications for,
706
00:50:01 --> 00:50:05
for example, therapies
against tumors.
707
00:50:05 --> 00:50:09
If you ask somebody, how do
you develop and how you judge
708
00:50:09 --> 00:50:13
the success of an anticancer
treatment? You talk to somebody
709
00:50:13 --> 00:50:17
like from the pharmaceutical
industry. And let's say that's easy.
710
00:50:17 --> 00:50:21
If you have a new drug, and
that drug reduces the mass of a
711
00:50:21 --> 00:50:26
tumor by 50%, that means that
you've done something really good.
712
00:50:26 --> 00:50:30
But let's look what's going on here.
If these cells are 99% of the tumor
713
00:50:30 --> 00:50:34
in terms of the mass and these
cells are 1% of the tumor,
714
00:50:34 --> 00:50:38
let's say you've invented a new drug
which wipes out all of these cells
715
00:50:38 --> 00:50:42
but doesn't touch these cells. The
bulk of the tumor has shrunk and
716
00:50:42 --> 00:50:46
everybody will say, eureka,
we've succeeded in curing
717
00:50:46 --> 00:50:50
cancer. But keep in mind that the
self-renewing capacity of the tumor
718
00:50:50 --> 00:50:53
rests in these cells. And
if these cells are allowed to
719
00:50:53 --> 00:50:57
survive, then they'll start
proliferating again and regenerate
720
00:50:57 --> 00:51:01
the entire tumor mass. And
you won't really know that you
721
00:51:01 --> 00:51:05
had any success because these cells
look like all the other tumor cells
722
00:51:05 --> 00:51:10
under the microscope. But
biologically, they're very
723
00:51:10 --> 00:51:14
different. And therefore,
the future of cancer therapy,
724
00:51:14 --> 00:51:19
and it will take five or ten years
to do this, has to begin to focus on
725
00:51:19 --> 00:51:23
getting rid of these self-renewing
stem cells which create this
726
00:51:23 --> 00:51:28
enormous regenerative
capacity on the part of tumors.
727
00:51:28 --> 00:51:32
See you next Monday.
Have a great vacation.
728
00:51:32 --> 51:37
Eat much turkey, and get some
exercise, and don't smoke.